Summary/Abstract: The characterization of myosin VI has generated a number of paradigm shifting concepts leading to the proposal that a subset of myosin classes form folded, inactive monomers until binding partner engagement induces activation. This binding additionally triggers dimerization (i.e. cargo-initiated dimerization) for at least three classes of myosin (VI, VIIA, and X), which are the focus of this proposal. While folding forms the basis of regulation of two-headed myosins from class II and V, the novel form of regulation found in class VI, VIIA, and X myosins can allow these myosins to diffuse throughout the cell as compact, folded monomers until they encounter their binding partners. Since most of the cellular functions served by these myosins are at the cell periphery, either in the cortical actin or in actin extensions, this may allow efficient delivery through dense actin networks. Two of the classes appear to be optimized for movement on bundles of actin (VIIA and X), while myosin VI traffics optimally on single actin filaments. Our most recent results suggest that all three classes share another feature, namely an anti-parallel coiled coil, which has not been described in any other myosin classes. This places the heads of the dimer in a geometry that may optimize their trafficking. We will probe the key features of these myosin classes with a combination of kinetic, single molecule, structural, and cell biological studies. In addition to sharing a common form of regulation, both class VI and VIIA myosins are involved in the assembly and maintenance of strereocilia of the cochlear hair cells. Mutations in these myosins lead to deafness. We will initiate the development of molecules that may be able to counter the impact of some of these deafness mutations.